Case Report
Elucidation of radiation-resistant clones by a serial study of intratumor heterogeneity before and after stereotactic radiotherapy in lung cancer Takahiro Nakagomi1, Taichiro Goto1, Yosuke Hirotsu2, Daichi Shikata1, Kenji Amemiya2, Toshio Oyama3, Hitoshi Mochizuki2, Masao Omata2,4 1
Department of General Thoracic Surgery; 2Genome Analysis Center, 3Department of Pathology, Yamanashi Central Hospital, Yamanashi, Japan;
4
University of Tokyo, Tokyo, Japan
Correspondence to: Taichiro Goto, MD, PhD. Department of General Thoracic Surgery, Yamanashi Central Hospital, Yamanashi, 400-8506, Japan. Email:
[email protected].
Abstract: Stereotactic radiotherapy (SRT) for inoperable stage I non-small cell lung cancer has shown promising results and is now an alternative therapy for this disease. Several reports have detailed changes in mutation profiles after treatment with chemotherapy; however, such changes after SRT for lung cancer have not been reported. A patient who received SRT for lung cancer developed local recurrence 9 months after treatment and underwent surgery in our department. Using bronchoscopically biopsied and surgically resected specimens, we performed targeted sequencing of 53 lung cancer-related genes and compared the tumor mutation profiles before and after SRT. Identical mutations were detected from tumor specimens collected before and after SRT, and the specimens were confirmed to be clonal. However, the number of mutations decreased after SRT, suggesting that it induced mutation selection. Analyses of the statistical inference of clonal population structure showed that this evolving heterogeneous genomic landscape may be caused by heterogeneous responsiveness to SRT. Keywords: Stereotactic radiotherapy (SRT); mutation; lung cancer; next-generation sequencing Submitted Mar 25, 2017. Accepted for publication May 23, 2017. doi: 10.21037/jtd.2017.06.02 View this article at: http://dx.doi.org/10.21037/jtd.2017.06.02
Introduction In recent years, stereotactic radiotherapy (SRT) has been effectively used to treat lung cancer, and it is steadily gaining acceptance as an alternative therapy to surgery for patients with physically inoperable conditions or those who refuse surgery (1-3). Meanwhile, there have also been sporadic reports of local recurrence and salvage surgery for local recurrence after SRT. Thus, the mechanism underlying tumor eradication by SRT warrants further investigation. In recent years, the detection of changes in mutation profiles after cancer treatment has attracted attention as a novel method for elucidating treatment efficacy (4). We encountered a patient who underwent surgery for local lung cancer recurrence after SRT. We investigated the therapeutic efficacy of SRT as a lung cancer treatment by
© Journal of Thoracic Disease. All rights reserved.
using molecular genetic testing to compare the lung cancer mutation profiles before and after SRT. Case presentation An 85-year-old woman visited our outpatient clinic for further examination of a right pulmonary nodule. Radiologic and bronchoscopic examinations revealed that the tumor was a cT1aN0M0 adenocarcinoma (Figure 1A). The patient refused surgery and chose to undergo SRT at a total radiation dose of 50 Gy in four fractions. Nine months after SRT, there appeared an area of consolidation that extended into the irradiated field (Figure 1B), and minimal pleural effusion also appeared in the right thorax. Radiologically, it was challenging to determine if the
jtd.amegroups.com
J Thorac Dis 2017;9(7):E598-E604
Journal of Thoracic Disease, Vol 9, No 7 July 2017
A
E599
B
Figure 1 Radiologic findings of the lung cancer. (A) A chest computed tomography scan showed a mass that is 16 mm in diameter with an irregular surface in the right lower lobe; (B) 9 months after SRT, an area of consolidation and minimal pleural effusion appeared.
consolidated area was local tumor recurrence or prolonged inflammation due to the radiation (Figure 1B). However, because carcinoembryonic antigen (CEA) levels were persistently elevated, we suspected tumor recurrence. When we informed the patient of the suspected diagnosis, she requested surgery; thus, a right lower lobectomy and lymph node dissections were performed (Figure 2A,B). During surgery, cytology revealed malignant pleural effusion, so the operation was considered “incomplete salvage surgery”. Histopathologically, most of the resected tumor had been replaced by elastofibrosis, but the lepidic growth pattern of the adenocarcinoma was present at the periphery of the elastic lesion (Figure 2C,D). The tumor cells had invaded the visceral pleura, but no lymph node metastases were found pathologically. After surgery, serum CEA levels immediately declined to within the normal range (Figure 3). The patient was prescribed gefitinib, an epidermal growth factor receptor (EGFR)-targeted drug, and has been followed up at our outpatient clinic without any sign of recurrence to date. Using a next-generation sequencer, we compared mutations in the specimens biopsied by bronchoscopic examination with those in the lung tumor surgical specimens. In both samples, cancer cells were collected from formalin-fixed paraffin-embedded tissues using laser capture microdissection. Tumor purity was about 80–90%. The total amount of DNA used for next-generation sequencing analysis was 1.62 ng of the biopsy specimens and 3.76 ng of the surgical specimens (Table S1). An assay system that could analyze the entire exon region of the 53 lung cancerrelated genes (68,046 amino acids) was developed in house, and the samples were subjected to next-generation sequencing (5-7). This assay system was used to identify mutations in the 53 significantly mutated genes, which were
© Journal of Thoracic Disease. All rights reserved.
identified from 408 patients with lung cancer by total exon analysis (8,9). In our study, mutations with an allele fraction ≥20% were deemed significant. Upon comparing mutations in the biopsy and surgical specimens, the same mutations in EGFR and MGA were detected, and interestingly, the positions and patterns of the base-pair substitutions for those mutations completely coincided (Figure 4). After SRT, the number of significant mutations decreased from three to two, and the number of insignificant mutations also decreased from eight to two (Figure 4). To determine whether radiotherapy changed the tumor cell composition, we performed PyClone analysis, which estimates the fraction of cancer cells harboring a mutation (Figure 5A,B) (10). Using pairwise correlation of mutations with cellular prevalence, the identified mutations were classified into four distinct clusters (Figure 5C). Of note, the cellular prevalence of clones with NOTCH1 mutations (p.Cys1374Tyr) was decreased after therapy (Figure 5D, cluster #2). In contrast, the cellular prevalence of tumor clones with EGFR and MGA mutations slightly increased after radiotherapy, indicating that these tumor clones remained after radiation therapy (Figure 5D, cluster #1). Together, these results suggest that radiosensitive or radioresistant tumor clones can coexist in a tumor (e.g., socalled intratumor heterogeneity); this heterogeneity may affect the response and resistance to radiotherapy. This study was approved by the Institutional Ethics Board of Yamanashi Central Hospital, and the patient provided written informed consent to participate. Discussion In this study, we used genetic testing to investigate the therapeutic efficacy of SRT for lung cancer by comparing
jtd.amegroups.com
J Thorac Dis 2017;9(7):E598-E604
E600
Nakagomi et al. Mutation selection of stereotactic radiotherapy for lung cancer
A
B
C
D
Figure 2 Pathological findings of the lung cancer. (A) A bronchoscopically biopsied specimen revealed invasive adenocarcinoma. The scale bar indicates 100 μm; (B) macroscopically, the resected specimen appeared as a grayish-white tumor just adjacent to the visceral pleura; (C) the majority of the resected tumor histopathologically consisted of necrotic or fibrous tissue, but the lepidic growth pattern of adenocarcinoma was observed at the periphery of the necrotic lesion (hematoxylin and eosin staining, 40×, low-power field); (D) the tumor cells were hobnail-like and lined the alveolar septa, displaying a lepidic growth pattern (hematoxylin and eosin staining, 100×, high-power field).
20 Surgery
18
Serum CEA level (ng/ml)
16 14 12
Gefitinib
SRT
10 8 6 4 2 0 -3 -2 -1 0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 months
Figure 3 Time course of serum CEA level. After SRT, serum CEA levels elevated until we performed surgery. Serum CEA levels normalized after surgery and gefitinib administration. The time of SRT, surgery, and the onset of gefitinib administration are indicated by the arrows. The horizontal axis denotes time, and each scale on the axis is set at 1-month intervals.
© Journal of Thoracic Disease. All rights reserved.
mutation profiles between specimens obtained before and after treatment. Although local recurrence indicated cancer progression, the number of mutations decreased, demonstrating the therapeutic efficacy of SRT. The patient, who had stage IA adenocarcinoma at the time of her initial examination, refused surgery and underwent SRT. A number of phase 2 trials demonstrated promising results from SRT, and local control rates are reportedly 80–95%, similar to surgical series (1-3). Particularly in patients ≥80 years of age, similar to the patient in our case study, definitive SRT for early-stage non-small-cell lung cancer is efficacious and safe (11,12). Two independent, randomized phase 3 trials that assessed SRT in patients with operable stage I non-small cell lung cancer closed early due to slow accrual; however, it was demonstrated that SRT could be an option for treating operable stage I non-small cell lung cancer (13). Additional randomized studies comparing SRT
jtd.amegroups.com
J Thorac Dis 2017;9(7):E598-E604
Journal of Thoracic Disease, Vol 9, No 7 July 2017
E601
Sample
Gene
Protein
Position
Ref.
Variance
AF (%)
BF biopsy
EGFR
p.E746-T751insVA
chr7:55242478
GAATTAAGAGAAGCAACA
GTTGCT
37
BF biopsy
NOTCH1
p.Cys1374Tyr
chr9:139400227
C
T
35
BF biopsy
MGA
p.Ile2540Met
chr15:42054436
A
G
34
BF biopsy
FGFR1
p.Val638Met
chr8:38273423
C
T
7
BF biopsy
NOTCH1
p.His1032Tyr
chr9:139403399
G
A
5
BF biopsy
ARID1B
p.Gln156Glu
chr6:157099529
C
G
5
BF biopsy
KMT2D
p.Pro634Leu
chr12:49445565
G
A
5
BF biopsy
AKT2
p.Asp441Asn
chr19:40740997
C
T
4
BF biopsy
NOTCH2
p.Met2467Ile
chr1:120457944
C
T
4
BF biopsy
RBM10
p.Glu751Lys
chrX:47044559
G
A
4
BF biopsy
U2AF1
p.Arg119Cys
chr21:44514892
G
A
4
Surgery
EGFR
p.E746-T751insVA
chr7:55242478
GAATTAAGAGAAGCAACA
GTTGCT
50
Surgery
MGA
p.Ile2540Met
chr15:42054436
A
G
48
Surgery
SOX2
p.Gln296Ter
chr3:181431034
C
T
5
Surgery
KMT2A
p.Gln2537Glu
chr11:118374216
C
G
4
Figure 4 Mutation profiles of lung cancer before and after SRT. Mutations with an allele fraction ≥20% are highlighted in red. Ref, reference; AF, allele fraction; BF, bronchoscopic fiber; SRT, stereotactic radiotherapy.
with surgery in patients with operable cancer are warranted. In this case, we originally intended to perform salvage surgery; however, because pleural dissemination was detected, we were unable to perform complete curative surgery. In current clinical practice, radiological and pathological difficulties in differentiating between inflammation and local recurrence after SRT are problematic. In our case study, because the tumor was predominantly composed of elastic tissue, a differential diagnosis might have been difficult to make even if a biopsy had been performed (14-16). Therefore, the development of cancer-specific diagnostic techniques, such as genetic testing or use of genomic biomarkers, is needed. In our study, EGFR and MGA mutations were identical in tumor specimens collected before and after SRT, suggesting that they belong to the same clone. Because of intratumor heterogeneity, tumors intrinsically contain various mutations (17,18). After exposure to radiation, it can be presumed that cancer cells with radiation-sensitive mutations are eradicated, and cancer cells with radiation-
© Journal of Thoracic Disease. All rights reserved.
resistant mutations survive. PyClone is a Bayesian clustering method for grouping sets of deeply sequenced somatic mutations into putative clonal clusters, while estimating their cellular prevalence and accounting for allelic imbalances introduced by segmental copy number changes and normal cell contamination (10). Thus, PyClone infers accurate clustering of mutations that co-occur in individual cells (10). In our study, PyClone analysis suggested that cells harboring NOTCH1 mutations were sensitive to radiation, whereas those harboring EGFR and MGA mutations were resistant to radiation. Recent studies suggest that activation of the NOTCH signaling pathway is related to radiation resistance in tumor cells (19-21). OncodriveROLE, a machine learning-based approach, indicated that the NOTCH1 p.Cys1374Tyr mutation, as detected in our study, is a “loss-of-function” mutation (22,23). Thus, in our case, loss-of-function in the NOTCH1 gene might have led to radiation sensitivity, resulting in the ability of cells harboring those mutations to be eradicated by radiotherapy.
jtd.amegroups.com
J Thorac Dis 2017;9(7):E598-E604
E602
Nakagomi et al. Mutation selection of stereotactic radiotherapy for lung cancer
A
B
EGFR:p.E746-T751insVA MGA:p.Ile2540Met NOTCH1:p.Cys1374Tyr FGFR1:p.Val638Met NOTCH1:p.His1032Tyr ARID1B:p.Gln156Glu KMT2D:p.Pro634Leu AKT2:p.Asp441Asn NOTCH2:p.Met2467Ile U2AF1:p.Arg119Cys RBM10:p.Glu751Lys
MGA:p.Ile2540Met EGFR:p.E746-T751insVA SOX2-OT|SOX2:|p.Gln296Ter KMT2A:p.Gln2537Glu 0.0
1.0
1.0 1.0
KMT2D:p.Pro634Leu ARID1B:p.Gln156Glu
0.9
NOTCH2:p.Met2467Ile
0.8
AKT2:p.Asp441Asn
0.7
U2AF1:p.Arg119Cys
D
1.0
0.8
0.8
Cluster 1 (n=2) Cluster 2 (n=1) Cluster 3 (n=2) Cluster 4 (n=8)
0.8
0.4 0.6 Cellular frequency
0.4 0.6 Cellular frequency
FGFR1:p.Val638Met
0.5
RBM10:p.Glu751Lys
0.4
MGA:p.Ile2540Met
0.6
0.6
NOTCH1:p.His1032Tyr
KMT2A:p.Gln2537Glu
0.1
SOX2-OT|SOX2:p.Gln296Ter SOX2-OT|SOX2:p.Gln296Ter
KMT2A:p.Gln2537Glu
NOTCH1:p.Cys1374Tyr
EGFR:p.E746-T751insVA
MGA:p.Ile2540Met
RBM10:p.Glu751Lys
FGFR1:p.Val638Met
U2AF1:p.Arg119Cys
NOTCH1:p.His1032Tyr
AKT2:p.Asp441Asn
ARID1B:p.Gln156Glu
NOTCH2:p.Met2467Ile
KMT2D:p.Pro634Leu
0.0
0.2
0.2
NOTCH1:p.Cys1374Tyr
0.4
0.3
EGFR:p.E746-T751insVA
0.0
C
0.2
Prevalence
0.0
0.2
Biopsy
Sample
Operation
Figure 5 Statistical inference of the clonal population structure. (A,B) Estimated cellular frequencies of the samples before and after SRT are shown as the distribution of posterior probabilities from the PyClone model; sample A: pre-radiation biopsy, sample B: post-radiation resection; (C) the correlation between all possible pairs of mutations with cellular prevalence was assessed to identify mutations that share a similar pattern of cellular prevalence. Unsupervised hierarchical clustering was used to group correlated mutations into four clusters, with the dendrogram threshold level used to establish clusters indicated on the y-axis (dotted line); (D) the inferred cellular prevalence changed after SRT based on the following cluster configuration: cluster 1, EGFR and MGA; cluster 2, NOTCH1; cluster 3, KMT2A and SOX2OT; cluster 4, other mutations.
We presume that this radiation-induced mutation selection resulted in decreased intratumor heterogeneity and a decreased number of detected mutations. Chemotherapy-induced changes in cancer mutation profiles have been reported, which can alter responsiveness to therapies (24-28). Here, we report that radiotherapy may also change lung cancer mutation profiles. However, because the broad clinical utility of our results has yet to be fully demonstrated, additional studies on similar cases treated with SRT are needed to validate our findings, as
© Journal of Thoracic Disease. All rights reserved.
well as to elucidate the genomic characteristics of radiation sensitivity and resistance. In addition, the development of new therapeutic strategies to eradicate patterns of mutation in cancer, such as a combination of molecular-targeted therapy and SRT, is needed. In conclusion, genetically identical mutations were detected in tumor specimens collected before and after SRT, and those specimens were confirmed to arise from the same clone. Meanwhile, because the number of mutations decreased after SRT, we estimated that SRT induced
jtd.amegroups.com
J Thorac Dis 2017;9(7):E598-E604
Journal of Thoracic Disease, Vol 9, No 7 July 2017
mutation selection. PyClone analysis showed that the therapeutic responsiveness of cancer to radiation varied among cells with different mutations, even in a single tumor. This condition is referred to as “heterogeneity in therapeutic responsiveness”, and may be responsible for the current difficulties in treating lung cancer. Acknowledgements The authors greatly appreciate Takuro Uchida, Hidetoshi Shigetomo, Yumi Kubota, and Ritsuko Yokouchi for their helpful scientific discussions.
E603
8.
9.
10.
11.
Footnote Conflicts of Interest: The authors have no conflicts of interest to declare. Informed Consent: Written informed consent was obtained from the patient for publication of this manuscript and all accompanying images. References 1. Nagata Y, Takayama K, Matsuo Y, et al. Clinical outcomes of a phase I/II study of 48 Gy of stereotactic body radiotherapy in 4 fractions for primary lung cancer using a stereotactic body frame. Int J Radiat Oncol Biol Phys 2005;63:1427-31. 2. Onishi H, Shirato H, Nagata Y, et al. Stereotactic body radiotherapy (SBRT) for operable stage I non-small-cell lung cancer: can SBRT be comparable to surgery? Int J Radiat Oncol Biol Phys 2011;81:1352-8. 3. Timmerman R, Paulus R, Galvin J, et al. Stereotactic body radiation therapy for inoperable early stage lung cancer. JAMA 2010;303:1070-6. 4. Lambrechts S, Smeets D, Moisse M, et al. Genetic heterogeneity after first-line chemotherapy in high-grade serous ovarian cancer. Eur J Cancer 2016;53:51-64. 5. Goto T, Hirotsu Y, Oyama T, et al. Analysis of tumorderived DNA in plasma and bone marrow fluid in lung cancer patients. Med Oncol 2016;33:29. 6. Goto T, Hirotsu Y, Mochizuki H, et al. Stepwise addition of genetic changes correlated with histological change from "well-differentiated" to "sarcomatoid" phenotypes: a case report. BMC Cancer 2017;17:65. 7. Goto T, Hirotsu Y, Mochizuki H, et al. Mutational analysis of multiple lung cancers: Discrimination between
© Journal of Thoracic Disease. All rights reserved.
12.
13.
14.
15.
16.
17.
18. 19.
20.
primary and metastatic lung cancers by genomic profile. Oncotarget 2017;8:31133-43. Cancer Genome Atlas Research N. Comprehensive genomic characterization of squamous cell lung cancers. Nature 2012;489:519-25. Cancer Genome Atlas Research N. Comprehensive molecular profiling of lung adenocarcinoma. Nature 2014;511:543-50. Roth A, Khattra J, Yap D, et al. PyClone: statistical inference of clonal population structure in cancer. Nat Methods 2014;11:396-8. Cassidy RJ, Patel PR, Zhang X, et al. Stereotactic Body Radiotherapy for Early-stage Non-small-cell Lung Cancer in Patients 80 Years and Older: A Multi-center Analysis. Clin Lung Cancer 2017. [Epub ahead of print]. Giuliani M, Hope A, Guckenberger M, et al. Stereotactic Body Radiation Therapy in Octo- and Nonagenarians for the Treatment of Early-Stage Lung Cancer. Int J Radiat Oncol Biol Phys 2017. [Epub ahead of print]. Chang JY, Senan S, Paul MA, et al. Stereotactic ablative radiotherapy versus lobectomy for operable stage I nonsmall-cell lung cancer: a pooled analysis of two randomised trials. Lancet Oncol 2015;16:630-7. Senthi S, Lagerwaard FJ, Haasbeek CJ, et al. Patterns of disease recurrence after stereotactic ablative radiotherapy for early stage non-small-cell lung cancer: a retrospective analysis. Lancet Oncol 2012;13:802-9. van den Berg LL, Klinkenberg TJ, Groen HJ, et al. Patterns of Recurrence and Survival after Surgery or Stereotactic Radiotherapy for Early Stage NSCLC. J Thorac Oncol 2015;10:826-31. Huang K, Dahele M, Senan S, et al. Radiographic changes after lung stereotactic ablative radiotherapy (SABR)--can we distinguish recurrence from fibrosis? A systematic review of the literature. Radiother Oncol 2012;102:335-42. Welch DR. Tumor Heterogeneity--A 'Contemporary Concept' Founded on Historical Insights and Predictions. Cancer Res 2016;76:4-6. Zellmer VR, Zhang S. Evolving concepts of tumor heterogeneity. Cell Biosci 2014;4:69. Shen Y, Chen H, Zhang J, et al. Increased Notch Signaling Enhances Radioresistance of Malignant Stromal Cells Induced by Glioma Stem/ Progenitor Cells. PLoS One 2015;10:e0142594. Yahyanejad S, King H, Iglesias VS, et al. NOTCH blockade combined with radiation therapy and temozolomide prolongs survival of orthotopic glioblastoma. Oncotarget 2016;7:41251-64.
jtd.amegroups.com
J Thorac Dis 2017;9(7):E598-E604
E604
Nakagomi et al. Mutation selection of stereotactic radiotherapy for lung cancer
21. Yahyanejad S, Theys J, Vooijs M. Targeting Notch to overcome radiation resistance. Oncotarget 2016;7:7610-28. 22. Schroeder MP, Rubio-Perez C, Tamborero D, et al. OncodriveROLE classifies cancer driver genes in loss of function and activating mode of action. Bioinformatics 2014;30:i549-55. 23. Southgate L, Sukalo M, Karountzos AS, et al. Haploinsufficiency of the NOTCH1 Receptor as a Cause of Adams-Oliver Syndrome With Variable Cardiac Anomalies. Circ Cardiovasc Genet 2015;8:572-81. 24. Morrissy AS, Garzia L, Shih DJ, et al. Divergent clonal selection dominates medulloblastoma at recurrence. Nature 2016;529:351-7. 25. Shaw AT, Friboulet L, Leshchiner I, et al. Resensitization
to Crizotinib by the Lorlatinib ALK Resistance Mutation L1198F. N Engl J Med 2016;374:54-61. 26. Song HN, Jung KS, Yoo KH, et al. Acquired C797S Mutation upon Treatment with a T790M-Specific ThirdGeneration EGFR Inhibitor (HM61713) in Non-Small Cell Lung Cancer. J Thorac Oncol 2016;11:e45-7. 27. Tan SH, Sapari NS, Miao H, et al. High-Throughput Mutation Profiling Changes before and 3 Weeks after Chemotherapy in Newly Diagnosed Breast Cancer Patients. PLoS One 2015;10:e0142466. 28. Diaz LA Jr, Williams RT, Wu J, et al. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature 2012;486:537-40.
Cite this article as: Nakagomi T, Goto T, Hirotsu Y, Shikata D, Amemiya K, Oyama T, Mochizuki H, Omata M. Elucidation of radiation-resistant clones by a serial study of intratumor heterogeneity before and after stereotactic radiotherapy in lung cancer. J Thorac Dis 2017;9(7):E598-E604. doi: 10.21037/ jtd.2017.06.02
© Journal of Thoracic Disease. All rights reserved.
jtd.amegroups.com
J Thorac Dis 2017;9(7):E598-E604
Supplementary
Table S1 DNA isolation and analysis Amount of DNA used for NGS (ng)
Sample
Dissected area (mm2)
Tumor purity (%)
Biopsy
15.7
80–90
30
0.27
1.62
Surgery
68.7
80–90
40
0.94
3.76
NGS, next generation sequencing.
Elution (µL) DNA Concentration (ng/µL)